Peptidomic Analysis of Skin Secretions of the Caribbean
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antibiotics Article Peptidomic Analysis of Skin Secretions of the Caribbean Frogs Leptodactylus insularum and Leptodactylus nesiotus (Leptodactylidae) Identifies an Ocellatin with Broad Spectrum Antimicrobial Activity Gervonne Barran 1, Jolanta Kolodziejek 2, Laurent Coquet 3 ,Jérôme Leprince 4 , Thierry Jouenne 3 , Norbert Nowotny 2,5 , J. Michael Conlon 6,* and Milena Mechkarska 1,* 1 Department of Life Sciences, Faculty of Science and Technology, The University of The West Indies, St. Augustine Campus, Trinidad and Tobago; [email protected] 2 Viral Zoonoses, Emerging and Vector-Borne Infections Group, Department of Pathobiology, Institute of Virology, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria; [email protected] (J.K.); [email protected] (N.N.) 3 CNRS UMR 6270, PISSARO, Institute for Research and Innovation in Biomedicine (IRIB), Normandy University, 76000 Rouen, France; [email protected] (L.C.); [email protected] (T.J.) 4 Inserm U1239, PRIMACEN, Institute for Research and Innovation in Biomedicine (IRIB), Normandy University, 76000 Rouen, France; [email protected] 5 Department of Basic Medical Sciences, College of Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai Helathcare City, P.O. Box 505055, Dubai, UAE 6 Diabetes Research Group, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK * Correspondence: [email protected] (J.M.C.); [email protected] (M.M.) Received: 21 August 2020; Accepted: 19 October 2020; Published: 20 October 2020 Abstract: Ocellatins are peptides produced in the skins of frogs belonging to the genus Leptodactylus that generally display weak antimicrobial activity against Gram-negative bacteria only. Peptidomic analysis of norepinephrine-stimulated skin secretions from Leptodactylus insularum Barbour 1906 and Leptodactylus nesiotus Heyer 1994, collected in the Icacos Peninsula, Trinidad, led to the purification and structural characterization of five ocellatin-related peptides from L. insularum (ocellatin-1I together with its (1–16) fragment, ocellatin-2I and its (1–16) fragment, and ocellatin-3I) and four ocellatins from L. nesiotus (ocellatin-1N, -2N, -3N, and -4N). While ocellatins-1I, -2I, and -1N showed a typically low antimicrobial potency against Gram-negative bacteria, ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2) was active against an antibiotic-resistant strain of Klebsiella pneumoniae and reference strains of Escherichia coli, K. pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium (minimum inhibitory concentrations (MICs) in the range 31.25–62.5 µM), and was the only peptide active against Gram-positive Staphylococcus aureus (MIC = 31.25 µM) and Enterococcus faecium (MIC = 62.5 µM). The therapeutic potential of ocellatin-3N is limited by its moderate hemolytic activity (LC50 = 98 µM) against mouse erythrocytes. The peptide represents a template for the design of long-acting, non-toxic, and broad-spectrum antimicrobial agents for targeting multidrug-resistant pathogens. Keywords: antibiotic resistance; antimicrobial peptides; frog skin secretions; hemolysis; Leptodactylus; norepinephrine stimulation; ocellatins; peptidomic analysis; phylogenetics; Trinidad 1. Introduction In an ongoing effort to address the global problem of antimicrobial resistance (AMR) and to promote the research and development of new antibiotics against emerging multidrug-resistant Antibiotics 2020, 9, 718; doi:10.3390/antibiotics9100718 www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, 718 2 of 15 bacteria, in 2017, the World Health Organization published a list of “priority pathogens” that cause increased morbidity and mortality and greatly impact healthcare costs [1]. Infections caused by antibiotic-resistant Gram-negative pathogens, such as extended-spectrum β-lactamase (ESBL) Escherichia coli and multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae, are of particular concern because the currently available treatment options are often ineffective and there are only a few antimicrobial drugs in the pipeline [2]. In order to address this crisis, the AMR Action Fund, encompassing more than 20 leading biopharmaceutical companies, was launched in July 2020, with the aim of bringing at least four new treatments to patients by 2030 [3]. AMR has been described as “a slow tsunami that threatens to undo a century of medical progress” [4]. Consequently, there is clearly a need to identify and develop new kinds of antimicrobial drugs with acceptable toxicological and pharmacokinetic properties that may be used to treat infections caused by multidrug-resistant pathogenic microorganisms. Anti-infective compounds based upon peptides are one such alternative to conventional antibiotics due to their rapid and non-specific mode of action, as well as their abilities to inhibit biofilm formation and act synergistically with established antibiotics [5]. Several frog species produce peptides with broad-spectrum antibacterial and antifungal activities that are also able to disrupt the plasma membrane of mammalian cells and more than 1000 such peptides have been described (reviewed in [6–11]). Although their precise biological role is incompletely understood, these peptides probably function as a component of the animal’s system of innate immunity, playing a role in the first-line defense against invading pathogens [12,13]. As peptides from different frog species may contain regions of structural similarity, they can be grouped into families for the purpose of classification. In addition, several of these peptides stimulate the production of pro- or anti-inflammatory cytokines by macrophages, inhibit viral replication, exert cytotoxicity towards cancer cells, and stimulate insulin release [14]. Consequently, it is more informative to refer to them as host-defence peptides (HDPs) than exclusively as antimicrobial peptides. Typically, each frog species has a “finger-print” arsenal of HDPs belonging to different families, whose primary structures can be used as a tool for elucidating the evolutionary history and complex phylogenetic relations of the different frog genera [15–17]. The genus Leptodactylus, or nest-building frogs, currently contains 78 species distributed from southern North America to central South America, including the West Indies [18]. Several frogs belonging to this genus have been shown to release structurally-related HDPs with antimicrobial activity into their skin secretions: Leptodactylus ocellatus [19–21], Leptodactylus fallax [22], Leptodactylus pentadactylus [23,24], Leptodactylus laticeps [25,26], Leptodactylus syphax [27], Leptodactylus validus [28], Leptodactylus labyrinthicus [29,30], Leptodactylus pustulatus [31], Leptodactylus latrans [32], and Leptodactylus vastus [33]. These peptides were initially named after the frog species from which they were first isolated, but they are now classified as ocellatins according to a generally accepted nomenclature [34]. In addition to the ocellatins, conformationally flexible glycine/leucine-rich plasticins, which lack antimicrobial activity, have been isolated from the skin secretions of L. pentadactylus [24] and L. laticeps [26]. As part of a program of investigation to systematically examine the frog species of Trinidad and Tobago for the presence of dermal biologically-active HDPs, this study involves an investigation of two Leptodactylus species that have not been studied previously: Leptodactylus insularum Barbour 1906 and Leptodactylus nesiotus Heyer 1994. The San Miguel Island frog L. insularum (also known as Barbour’s Thin-Toed frog) is a medium to large frog (snout-vent-length (SVL) for sub-adults/adults is in the range of 51–83 mm). Previously classified as L. pentadactylus and Leptodactylus bolivianus [35], L. insularum is found from Costa Rica to Trinidad, but it has not been recorded in Tobago. In Trinidad, this species has been found at low elevations in the central and southeast parts of the island. The preferred habitats of the frog include forested areas and swamp margins. Because of its wide distribution range, tolerance of varied habitats, and presumably large populations, L. insularum is classified by the International Union for Conservation and Nature (IUNC) Red List of Threatened Species as a species of Least Concern. The Trinidad Thin-Toed frog L. nesiotus is a small terrestrial frog (adult males SVL 33 mm Antibiotics 2020, 9, 718 3 of 15 and sub-adults SVL 21–28 mm). This species was considered to be endemic to Trinidad until Jairam and Fouquet [36] reported, for the first time, its presence on the South American continent in Guyana, Suriname, and French Guiana. L. nesiotus can be found inhabiting open and swampy areas of the Icacos Peninsula, as well as many scattered locations in southern Trinidad [35]. L. nesiotus is listed as a Vulnerable Species by the IUCN. Both frog species investigated in this study were identified on the basis of their audio calls and characteristic morphologym as described by Murphy et al. [35]. An injection of norepinephrine was used to stimulate the release of HDPs into skin secretions, which were purified and characterized by reversed-phase HPLC in combination with MALDI-TOF mass spectrometry and automated Edman degradation. The peptides isolated from L. insularum are denoted by I and those isolated from L. nesiotus by N. Peptides from paralogous genes are differentiated by numerals, e.g.,